Currently, typical methods of assaying mutations in the nucleotide sequence of DNA include Single-strand conformation polymorphism (SSCP) and DNA sequencing.
The SSCP method, developed by M. Orita et al in 1989, is a method to analyze polymorphisms by utilizaing the fact that single-strand DNA adopts a higher-order structure dependent on the nucleotide sequence, and the structure are detected as differences in mobility by polyacrylamide gel electrophoresis. It is also called PCR-SSCP since DNA fragments amplified by PCR are often employed.
Because this method uses electrophoresis, however, it cannot identify the location and the type of nucleotide of the mutation. In addition, SSCP is non-qunatitative analysis and difficult to rapidly analyze many samples at the same time.
There are several different kinds of DNA sequencing methods, but the fundamental one is Sanger's method, developed in 1975. In this method, DNA fragment of interest is converted to a single strand, and the complementary strand against to a fluorescent labeled primer is elongated by DNA polymerase in the presence of following reagents; fluorescent labeled primers that recognizes the 5′ upstream sequence from the site of interest, four types of deoxynucleotide (dNTP) including adenine (dATP), thymine (dTTP), guanine (dGTP) and cytosine (dCTP), and one type of dideoxynucleotide. DNA elongation will progress when dNTP (a constituent of DNA) is incorporated until a ddNTP is incorporated. Because of a hydroxyl group added at the binding site of the next nucleotide, the polymerization reaction is blocked, and the DNA can no longer be elongated. As a result, multiple DNA fragments are produced with specific bases at their ends. In this method, four different samples are produced using four types of ddNTP, and the nucleotide sequence can be analyzed by performing electrophoresis and reading the resulting DNA bands in sequence.
However, although this method is suited to analyzing a broad range of nucleotide sequences from DNA fragments, it is not convenient enough when the object of study is a mutation at a specific site.
Dye-labeled oligonucleotide ligation (DOL) is another method of detecting nucleotide mutations. DOL utilizes the binding of two oligonucletides with DNA ligase. Following two types of oligonucleotides are prepared; an oligo probe complementary to a sequence labeled with fluorescein at the 5′ end and ending one nucleotide upstream from the SNP site, and an oligo probe fluorescently labeled at the 3′ end which includes the SNP region and is complementary to the sequence downstream therefrom. According to the differences in the complementary nucleotide of the SNP region, the second probe is labeled with different fluorescent dye such as ROX or TAMRA. When these probes and DNA ligase are added in PCR reaction mixture, the number of probes joined by ligase is increased as the PCR reaction progress. Since the only joined probes are those which are complementary to the SNP region, excitation of fluorescein allows the SNP to be determined based on what fluorescent wavelength emitted as a result of fluorescence resonance energy transfer (FRET). DOL assay is extremely simple because all reactions are performed simultaneously in one tube, but unfortunately it is not suitable for multiple specimens processing.
Another ligation method which has been developed for detecting nucleotide mutations is Genespector (trade name; Variom Biotechnology AG analysis kit). In this method, probes are fixed on a solid surface and the detection is achieved by a ligation reaction following a hybridization reaction using the DNA PCR product, a signal probe and an allele-specific detection probe.
However, this method has problems of detection sensitivity of the nucleotide mutation and speed of the mutation analysis.
Hereinafter, an invention to determine and assay a nucleotide type at a specific location in DNA or RNA, and rapidly analyzes a variety of mutations including single nucleotide polymorphism, repeated nucleotide sequence mutations, nucleotide deletion mutations, nucleotide insertion mutations and translocation mutations is presented.